Detection of PD‑L1 expression and epithelial‑mesenchymal transition of circulating tumor cells in non‑small cell lung cancer

Abstract

The present study aimed to assess the roles of peripheral circulating tumor cell (CTC) count, CTC subtypes and programmed death ligand 1 (PD-L1) expression in the clinical staging and prognosis of patients with non-small cell lung cancer (NSCLC). A total of 100 patients with NSCLC with available tumor tissues were enrolled in the present study, and 7.5 ml peripheral blood was collected. Patients were divided into PD-L1-positive and PD-L1-negative groups according to PD-L1 immunohistochemical staining. Peripheral blood samples from both groups were analyzed to determine the CTC count, epithelial-type CTCs (E-CTCs), mesenchymal-type CTCs (M-CTCs) and PD-L1 expression. Clinical data were collected, and patients were followed up for a maximum of 36 months, with patient death as the endpoint event. Patients with PD-L1-positive tumors had a worse prognosis compared with those with PD-L1-negative tumors (P=0.045). The PD-L1-positive group exhibited significantly higher numbers of CTCs and M-CTCs compared with the PD-L1-negative group (P≤0.05). However, the number of E-CTCs did not differ significantly between the two groups (P>0.05). PD-L1-positive patients with higher CTC and M-CTC counts had relatively poorer prognoses (P≤0.05), while the number of E-CTCs had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the CTC count (P≤0.05), while E-CTC and M-CTC counts did not significantly differ between the two groups (P>0.05). The PD-L1-positive group exhibited a significant increase in the number of PD-L1⁺ CTCs and PD-L1⁺ M-CTCs compared with the PD-L1-negative group (P≤0.05), while PD-L1⁺ E-CTC counts did not differ significantly between the two groups (P>0.05). The PD-L1-positive patients with a higher number of PD-L1⁺ CTCs and PD-L1⁺ M-CTCs had relatively poorer prognoses (P≤0.05), while the PD-L1⁺ E-CTC count had no significant effect on prognosis (P>0.05). Compared with the early-stage NSCLC group, the late-stage NSCLC group exhibited a significant increase in the number of PD-L1⁺ CTCs and PD-L1⁺ M-CTCs (P≤0.05), while PD-L1⁺ E-CTC counts did not significantly differ between the two groups (P>0.05). Based on univariate and multivariate analyses, the number of PD-L1⁺ M-CTCs was identified as an independent prognostic factor for NSCLC. In conclusion, the presence of CTCs in peripheral blood, particularly PD-L1⁺ M-CTC subtype, indicated poorer clinical staging and prognosis in patients with NSCLC. These findings suggested that CTCs, specifically the PD-L1⁺ M-CTC subtype, could serve as a monitoring indicator for the clinical staging and prognosis of patients with NSCLC.

Keywords: circulating tumor cell; epithelial-mesenchymal transition; mesenchymal; non-small cell lung cancer; programmed death ligand 1.

Figure 1

Expression of PD-L1 in tumor tissues and prognosis of patients with NSCLC. (A) PD-L1-positive and (B) PD-L1-negative IHC staining in tumor tissues of patients with NSCLC. (C) PD-L1-positive and (D) PD-L1-negative HE staining in tumor tissues from patients with NSCLC (Scale bar, 100 µm). (E) Kaplan-Meier overall survival probability analysis of PD-L1-positive and PD-L1-negative patients. HE, hematoxylin and eosin; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1.

Figure 2

Quantification of CTCs in patients with NSCLC and association with prognosis. (A) Representative images of cells stained with DAPI, CK-FITC and CD45-PE to identify CTCs in peripheral circulation of patients with NSCLC by immunofluorescence staining. Scale bar, 10 µm. (B) Kaplan-Meier analysis of OS in ≤5 CTC and >5 CTC groups of PD-L1-positive and PD-L1-negative patients. Quantification of CTCs, E-CTCs and M-CTCs in peripheral circulation of (C) PD-L1-positive and PD-L1-negative groups and (D) stage I-II and stage III-IV groups. Data are presented as the mean ± SD. Kaplan-Meier analysis of survival probability in (E) The OS of M-CTC on the PD-L1⁺-group and PD-L1^--group; (F) The OS of E-CTC on the PD-L1⁺-group and PD-L1^--group. ^*P<0.05, ^***P<0.001. CK, cytokeratin; CTC, circulating tumor cell; E-CTC, epithelial-type CTC; M-CTC, mesenchymal-type CTC; ns, not significant; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1; PE, phycoerythrin; WF, white field.

Figure 3

Quantification of PD-L1+ CTCs and CTC subtypes in patients with NSCLC. (A) Representative images of cells stained with DAPI, pcytokeratins (CK-FITC), CD45-PE and PD-L1 (AF647) to identify PD-L1⁺ CTCs in peripheral circulation of patients with NSCLC by immunofluorescence staining. Scale bar, 10 µm. (B) Kaplan-Meier analysis of survival probability in ≤5 PD-L1⁺ CTCs and >5 PD-L1⁺ CTCs groups of PD-L1-positive patients, and ≤5 CTCs and >5 CTCs groups of PD-L1-negative patients. Quantification of PD-L1⁺ CTCs, PD-L1⁺ E-CTCs and PD-L1⁺ M-CTCs in peripheral circulation of (C) PD-L1-positive and PD-L1-negative groups and (D) stage I-II and stage III-IV groups. Data are presented as the mean ± SD. Kaplan-Meier analysis of survival probability in (E) PD-L1⁺ M-CTC on the PD-L1⁺-group and PD-L1^--group; (F) OS of PD-L1⁺ E-CTC on the PD-L1⁺-group and PD-L1^--group. ^*P<0.05, ^***P<0.001. CK, cytokeratin; CTC, circulating tumor cell; E-CTC, epithelial-type CTC; M-CTC, mesenchymal-type CTC; ns, not significant; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1; PE, phycoerythrin; WF, white field.